The present invention relates generally to materials and methods for use in immunological procedures for quantitative detection and isolation of the hormone erythropoietin in fluid samples. More specifically, the invention relates to a monoclonal anti-erythropoietin antibody produced by a novel tumor cell line A.T.C.C. HB8209. The antibody is immunologically reactive with a low molecular weight polypeptide having an amino acid sequence substantially duplicative of a sequence extant in erythropoietin. The antibody is useful in diagnostic assays on human fluids and in procedures for affinity purification and isolation of human erythropoietin.
Erythropoiesis, the production of red blood cells, occurs continuously throughout the human life span to offset cell destruction. Erythropoiesis is a very precisely controlled physiological mechanism enabling sufficient numbers of red blood cells to be available in the blood for proper tissue oxygenation, but not so many that the cells would impede circulation. The formation of red blood cells occurs in the bone marrow and is under the control of the hormone, erythropoietin.
Erythropoietin, an acidic glycoprotein of approximately 34,000 molecular weight, may occur in three forms: .alpha., .beta., and asialo. The .alpha. and .beta. forms differ slightly in carbohydrate components, but have the same potency, biological activity and molecular weight. The asialo form is an .alpha. or .beta. form with the terminal carbohydrate (sialic acid) removed. Erythropoietin is present in very low concentrations in plasma when the body is in a healthy state wherein tissues receive sufficient oxygenation from the existing number of erythrocytes. This normal low concentration is enough to stimulate replacement of red blood cells which are lost normally through aging.
The amount of erythropoietin in the circulation is increased under conditions of hypoxia when the number of red blood cells in the circulation is reduced. Hypoxia may be caused by loss of large amounts of blood through hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged unconsciousness, or various forms of anemia. In response to tissues undergoing hypoxic stress, erythropoietin will increase red blood cell production by stimulating the conversion of primitive precursor cells in the bone marrow into pro-erythroblasts, bone marrow cells which subsequently mature, synthesize hemoglobin and are released into the circulation as red blood cells. When the number of red blood cells in circulation is greater than needed for normal tissue oxygen requirements, erythropoietin in circulation is decreased.
Because erythropoietin is essential in the process of red blood cell formation, the hormone has potential useful application in both the diagnosis and the treatment of blood disorders characterized by low or defective red blood cell production. Prior attempts to obtain erythropoietin in good yield from plasma or urine, however, have proven relatively unsuccessful. Complicated and sophisticated laboratory techniques are necessary and generally result in the collection of very small amounts of impure and unstable extracts containing erythropoietin.
U.S. Pat. No. 3,033,753 describes a method for partially purifying erythropoietin from sheep blood plasma which provides low yields of a crude solid extract containing erythropoietin.
Initial attempts to isolate erythropoietin from urine yielded unstable, biologically inactive preparations of the hormone. U.S. Pat. No. 3,865,801 describes a method of stabilizing the biological activity of a crude substance containing erythropoietin recovered from urine. The resulting crude preparation containing erythropoietin purportedly retains 90% of erythropoietin activity, and is stable.
Another method of purifying human erythropoietin from urine of patients with aplastic anemia is described in Miyake, et al., J. Biol. Chem., Vol. 252, No. 15 (Aug. 10, 1977), pp. 5558-5564. This seven-step procedure includes ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, and yields a crude erythropoietin preparation with a potency of 70,400 units/mg of protein in 21% yield. The purified hormone has a single electrophoretic component in polyacrylamide gels at pH 9, in the presence of sodium dodecyl sulfate at pH 7, and in the presence of Triton X-100 at pH 6. Two fractions of the same potency and molecular size, by sodium dodecyl sulfate gel electrophoresis, but differing slightly in mobility at pH 9, are obtained at the last step of fractionation.
Other techniques utilized to obtain purified erythropoietin involve immunological procedures. A polyclonal, serum-derived antibody directed against erythropoietin is developed by injecting an animal, preferably a rat or rabbit, with human erythropoietin. The injected human erythropoietin is recognized as a foreign antigenic substance by the immune system of the animal and elicits production of antibodies against the antigen. Differing cells responding to stimulation by the antigenic substance produce and release into circulation antibodies slightly different from those produced by other responding cells. The antibody activity remains in the serum of the animal when its blood is extracted. While unpurified serum or antibody preparations purified as a serum immunoglobulin G fraction may then be used in assays to detect and complex with human erythropoietin, the materials suffer from a major disadvantage. This serum antibody, composed of all the different antibodies produced by individual cells, is polyclonal in nature and will complex with components in crude extracts other than erythropoietin alone.
Of interest to the background of the present invention are recent advances in the art of developing continuous cultures of cells capable of producing a single species of antibody which is specifically immunologically reactive with a single antigenic determinant of a selected antigen. See, generally, Chisholm, High Technology, Vol. 3, No. 1, 57-63 (1983). Attempts have been made to employ cell fusion and hybridization techniques to develop "monoclonal" antibodies to erythropoietin and to employ these antibodies in the isolation and quantitative detection of human erythropoietin. As one example, a report of the successful development of mouse-mouse hybridoma cell lines secreting monoclonal antibodies to human erythropoietin appeared in abstract form in Abstract No. 1463 of Fed. Proc., 41, 520 (1982). As another example, a detailed description of the preparation and use of a monoclonal, anti-erythropoietin antibody appears in Weiss, et al., P.N.A.S. (U.S.A.), 79, 5465-5469 (1982).
To the extent that the two above-noted publications may provide access to monoclonal antibodies useful in the efficient isolation of erythropoietin by immunological affinity purification and the accurate quantitative detection of erythropoietin by immunobinding assays, such purifications and assays will nonetheless be prey to the problems which typically beset other immunological procedures relating to other biologically active proteinaceous materials. As one example, a common problem in most procedures for affinity purification of proteinaceous materials (whether monoclonal or polyclonal antibodies are used) is that harshly acidic or basic solutions are needed to elute the protein bound to the immobilized antibody. Frequently elution conditions diminish or destroy the biological activity of the material to be isolated. As another example, where immunobinding assays are employed in quantitative detection of a proteinaceous material in a fluid sample, the amount of material present in the sample is usually determined by comparison of the extent of an immunobinding reaction of the antibody to a "standard" solution including a fixed quantity of the same proteinaceous material in pure form. One example of such an assay is a competition reaction wherein proteinaceous material competes for the antibody in the presence of a "standard" solution including a fixed quantity of the same proteinaceous material in pure form. Quantification of the competition reaction is made by comparison to the extent of reaction of the antibody with increasing quantities of pure standard. Where, as in the case of erythropoietin, the material to be assayed is a rather labile substance which is easily destroyed in processes (including radiolabelling processes) for generating and storing standard solutions, the accuracy of the entire assay system can be severely compromised.
Also of interest to the background of the invention are reports of the immunological activity of synthetic peptides which substantially duplicate the amino acid sequence extant in naturally-occurring proteins, glycoproteins and nucleoproteins. More specifically, relatively low molecular weight polypeptides have been shown to participate in immune reactions which are similar in duration and extent to the immune reactions of physiologically significant proteins such as viral antigens, polypeptide hormones and the like. Included among the immune reactions of such polypeptides is the provocation of the formation of specific antibodies in immunologically active animals. See, e.g., Lerner, et al., Cell, 23, 309-310 (1981); Ross, et al., Nature, 294, 654-656 (1981); Walter, et al., P.N.A.S. (U.S.A.), 77, 5197-5200 (1980); Lerner, et al., P.N.A.S. (U.S.A.), 78, 3403-3407 (1981); Walter, et al., P.N.A.S. (U.S.A), 78, 4882-4886 (1981); Wong, et al., P.N.A.S. (U.S.A.), 78, 7412-7416 (1981); Green, et al., Cell, 28, 477-487 (1982); Nigg, et al., P.N.A.S. (U.S.A.), 79, 5322-5326 (1982); Baron, et al., Cell, 28, 395-404 (1982); Dreesman, et al., Nature, 295, 158-160 (1982); and Lerner, Scientific American, 248, No. 2, 66-74 (1983). The above studies relate, of course, to amino acid sequences of proteins other than erythropoietin, a substance for which no substantial amino acid sequence information has been published.
Despite recent substantial advances in the art, there continues to exist a substantial need in the art for further new methods and materials useful in obtaining large quantities of pure, biologically active erythropoietin from plasma or urine and methods and materials useful in the accurate quantitative detection of erythropoietin in human fluid samples.